Antibodies are normally synthesized by lymphoid cells derived from B lymphocytes of bone marrow. Lymphocytes derived from the same clone produce immunoglobulin of a single amino acid sequence. Lymphocytes cannot be directly cultured over long periods of time to produce substantial amounts of their specific antibody. However, Kohler et al. (1975) Nature 256:495-497, demonstrated that a process of somatic cell fusion, specifically between a lymphocyte and a myeloma cell, could yield hybrid cells which grow in culture and produce a specific antibody called a "monoclonal antibody" (hereinafter also referred to as "MAB"). The resulting hybrid cell was called a "hybridoma". A monoclonal antibody belongs to a group of antibodies whose population is substantially homogeneous, i.e. the individual molecules of the antibody population are identical except for naturally occurring mutations. Myeloma cells are lymphocyte tumor cells which, depending upon the cell strain, frequently produce an antibody themselves, although "non-producing" strains are known.
The development of monoclonal antibody technology has provided an enormous opportunity for science and medicine in implementing research, diagnosis and therapy. Monoclonal antibodies are used in radioimmunoassays, enzyme-linked immunosorbent assays, immunocytopathology, and flow cytometry for in vitro diagnosis, and in vivo for diagnosis and immunotherapy of human disease. Waldmann, T. A. (1991) Science 252:1657-1662. In particular, monoclonal antibodies have been widely applied to the diagnosis and therapy of cancer, wherein it is desirable to target malignant lesions while avoiding normal tissue. See, e.g., U.S. Pat. Nos. 4,753,894 to Frankel, et al.; 4,938,948 to Ring et al.; and 4,956,453 to Bjorn et al.
For a number of practical and economic reasons, most clinical applications have been based on murine antihuman monoclonal antibodies. Murine antibodies can be raised against molecules which are particularly associated with neoplastic cells using techniques known in the art. In this regard, tumor cells express increased numbers of various receptors for molecules which augment their proliferation (Goustin et al. (1986) Cancer Res. 46:1015-1029), many of which receptors are the products of oncogenes (Cline et al. (1984) Ann Intern Med. 101:223-228). Thus, a number of monoclonal antibodies directed against receptors for transferrin (Taetle et al. (1987) Cancer Res. 47:2040-2044 and Sauvage et al. (1987) Cancer Res. 47:747-753), interleukin-2 (Waldmann, T. A. (1986) Science 232:727-732 and Wong, et al. (1987) J. Exp Med. 166:1055-1069), and epidermal growth factor (Masui et al. (1984) Cancer Res. 44:1002-1007, Sato et al. (1983) Mol Biol Med. 1:511-529, and Rodeck et al. (1987) Cancer Res. 57:3692-3696) have been described. Although such molecules have antigen binding specificities of significant therapeutic value, the use of such murine antibodies in the treatment of human neoplastic disease has been limited since those molecules are immunogenic to the human immune system.
A number of investigators have used monoclonal antibodies as carriers of cytotoxic substances in attempts to selectively direct those agents to malignant tissue. In this manner, radioisotopes, natural toxins, chemotherapy agents, or other substances (such as biological response modifiers) are chemically linked or conjugated to a monoclonal antibody to form "immunoconjugates" and "immunotoxins." More particularly, a number of monoclonal antibodies have been conjugated to toxins such as ricin, abrin, diphtheria toxin and Pseudomonas exotoxin or to enzymatically active portions (A chains) thereof via heterobifunctional agents. See, e.g., U.S. Pat. No. 4,753,894 to Frankel et al.; Nevelle, et al. (1982) Immunol Rev 62:75-91; Ross et al. (1980) European J Biochem 104; Vitteta et al. (1982) Immunol Rev 62:158-183; Raso et al. (1982) Cancer Res 42:457-464, and Trowbridge et al. (1981) Nature 294:171-173. However, several factors have limited therapy using immunoconjugates or immunotoxins, particularly the production of human antimurine antibodies which greatly lowers the therapeutic index associated with those agents.
Since the mid-1970's, there have been numerous reports of murine monoclonal antibodies that interact with human breast cancer associated antigens. In these reported studies, mice were immunized and boosted with human milk fat globule proteins, breast cancer cell lines or breast cancer membrane extracts. Immune splenocytes were fused with mouse myeloma cells and hybridomas were selected based on some specificity of the culture media for breast or breast cancer antigens. Taylor-Papadimitriou, et al. (1981) Int. J. Cancer 28:17-21; Yuan et al. (1982) JNCI 68:719-728; Ciocca et al. (1982) Cancer Res. 42:4256-4258.
Because of the immunogenicity problems associated with the therapeutic use of murine antibody molecules, a number of chimeric antibodies composed of human and non-human amino acid sequences have been proposed. Particularly, hybrid antibody molecules having variable regions derived from, for example, a murine immunoglobulin fused to constant regions derived from a human immunoglobulin have been described. See e.g., U.S. Pat. No. 4,816,567; Winter et al. (1991) Nature 349:293-299; and Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224. Further, since constant regions are not required for antigen recognition or binding, antibody fragments such as F(ab), F(ab').sub.2 and Fv which do not comprise the Fc portion have been indicated as useful in radioimmunodetection, or as candidates for conjugation to a large toxin subunit such as ricin A-chain to provide a less immunogenic immunotoxin with an appreciable serum half-life. Dillman, R. O. (1989) Ann Intern Med. 111(7):592-603.
A number of recombinant or biosynthetic molecules comprising rodent antigen-binding sites have been described. Particularly, molecules having rodent antigen-binding sites built directly onto human antibodies by grafting only the rodent binding site, rather than the entire variable domain, into human immunoglobulin heavy and light chain domains have been described. See, e.g., Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536. Molecules having an antigen-binding site wherein at least one of the complementarity determining regions (CDRS) of the variable domain is derived from a murine monoclonal antibody and the remaining immunoglobulin-derived parts of the molecule are derived from human immunoglobulin have been described in U.K Patent Publication No. GB 2,276,169, published Sep. 21, 1994. A number of single chain antigen-binding site polypeptides and single chain Fv (sFv) molecules have also been described. See, e.g., U.S. Pat. Nos. 5,132,405 and 5,091,513 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.